The relationships between gut microbiota and animal health are an important consideration that are increasingly influential in the management of wild and ex situ endangered species, such as the cheetah (Acinonyx jubatus). To better understand these relationships, fresh fecal samples are currently required as a non-invasive alternative for the gut microbiome. Unfortunately, fresh samples are challenging to collect, especially in the wild. This study had two main aims: 1) to determine the optimal collection time point for cheetah feces after deposit in their native environment of Namibia as a guide for wild cheetah fecal microbiome studies; and 2) to characterize the cheetah fecal microbiota between two ex situ cheetah populations from two locations (Front Royal, VA, USA and Otjiwarongo, Namibia), which also consume different diets (commercial diet + carcass supplements and carcass only, respectively). Fresh fecal samples were collected from 16 cheetahs (Virginia, n = 8; Namibia n = 8) and analyzed for bacterial community diversity and composition using 16S rRNA gene amplicon sequencing. First, we found no difference in bacterial richness, diversity, or community composition from fresh fecal material compared to subsequent samples of decomposing fecals over a four-day sampling period in Namibia. Second, fresh cheetah samples in semi-wild conditions in Namibia had higher number of bacterial taxa, more phylogenetically diverse bacteria and contained compositionally distinct microbiomes (Bray-Curtis dissimilarity, Jaccard, and unweighted UniFrac measures) from cheetahs in zoo-managed conditions in the USA. We also found few differences in predictive functions of the fecal microbiota between the populations, where only one disease-related pathway was higher in the USA samples. Overall, our findings suggest that in dry season conditions (no recorded rainfall) in Namibia, fecals may be usable for up to three days after defecation for microbial ecology studies. We provide observational data on how to identify fecals within four days of being voided (see supplemental photos). There are major demographic and dietary differences between ex situ Namibian and USA populations, and we suggest further investigation into the influence of diet and population on the gut microbiota and health of cheetahs.